The increased number of cancer cases reported in the United States, and, indeed, around the world, is a major concern. Currently there are only a handful of treatments available for specific types of cancer, and these provide no absolute guarantee of success. In order to be most effective, these treatments require not only an early detection of the malignancy, but a reliable assessment of the severity of the malignancy.
The incidence of breast cancer, a leading cause of death in women, has been gradually increasing in the United States over the last thirty years. In 1997, it was estimated that 181,000 new cases were reported in the U.S., and that 44,000 people would die of breast cancer (Parker et al, 1997, CA Cancer J. Clin. 47:5-27; Chu et al, 1996, J. Nat. Cancer Inst. 88:1571-1579). While the pathogenesis of breast cancer is unclear, transformation of normal breast epithelium to a malignant phenotype may be the result of genetic factors, especially in women under 30 (Miki et al., 1994, Science, 266:66-71). The discovery and characterization of BRCA1 and BRCA2 has recently expanded our knowledge of genetic factors that can contribute to familial breast cancer. Germ-line mutations within these two loci are associated with a 50 to 85% lifetime risk of breast and/or ovarian cancer (Casey, 1997, Curr. Opin. Oncol. 9:88-93; Marcus et al., 1996, Cancer 77:697-709). However, it is likely that other, non-genetic factors also have a significant effect on the etiology of the disease. Regardless of its origin, breast cancer morbidity and mortality increases significantly if it is not detected early in its progression. Thus, considerable effort has focused on the early detection of cellular transformation and tumor formation in breast tissue.
Currently, the principal manner of identifying breast cancer is through detection of the presence of dense tumorous tissue. This may be accomplished to varying degrees of effectiveness by direct examination of the outside of the breast, or through mammography or other X-ray imaging methods (Jatoi, 1999, Am. J. Surg. 177:518-524). The latter approach is not without considerable cost, however. Every time a mammogram is taken, the patient incurs a small risk of having a breast tumor induced by the ionizing properties of the radiation used during the test. In addition, the process is expensive and the subjective interpretations of a technician can lead to imprecision, e.g., one study showed major clinical disagreements for about one-third of a set of mammograms that were interpreted individually by a surveyed group of radiologists. Moreover, many women find that undergoing a mammogram is a painful experience. Accordingly, the National Cancer Institute has not recommended mammograms for women under fifty years of age, since this group is not as likely to develop breast cancers as are older women. It is compelling to note, however, that while only about 22% of breast cancers occur in women under fifty, data suggests that breast cancer is more aggressive in pre-menopausal women.
Human breast tumors are diverse in their natural history and in the responsiveness to treatments. Variation in transcriptional programs accounts for much of the biological diversity of human cells and tumors. In each cell, signal transduction and regulatory systems transduce information from the cell's identity to its environmental status, thereby controlling the level of expression of every gene in the genome. (Perou, C. et al., (2000) Nature 406:747-752).
Human breast tumors are histologically complex tissues, containing a variety of cell types in addition to the carcinoma cells. Two distinct types of epithelial cell are found in the human mammary gland: basal (and/or myoepithelial) cells and luminal epithelial cells. These two cell types are conveniently distinguished immunohistochemically; basal epithelial cells can be stained with antibodies to keratin 5/6, whereas luminal epithelial cells stain with antibodies against keratins 8/18. (Perou et al., supra)
Gene expression information generated by DNA microarray analysis of human tumors can provide molecular phentotyping that can identify distinct tumor classifications not evident by traditional histopathological methods. (West, M. et al., (2001) PNAS 2001 98: 11462-11467). The analysis of gene expression represents an indirect measure of the genetic alterations in tumors because, in most instances, these alterations affect gene regulatory pathways. Given the tremendous complexity that can be scored by measuring gene expression with DNA microarrays, together with the absence of bias in assumptions as to what type of pathway might be affected in a particular tumor, the analysis of gene expression profiles offers the potential to impact clinical decision-making based on more precise determinations of tumor cell phenotypes. (West et al., supra).
Currently, about 100 critical genes in growth stimulatory pathways have been identified as potential targets for development as therapeutic agents. Human epidermal growth factor receptor-2 (“HER2”) is a member of a class of molecules in this pathway, called growth factor receptors. Following discovery of the HER2 probe in 1987, it was determined that 25% to 30% of women with breast cancer have amplification of this gene. Although expression does not always follow gene amplification, HER2 expression was found to directly correlate to its degree of amplification, such that amplification results in overexpression in 95% of cases. When HER2 overexpression occurs, at some point between premalignant and preinvasive disease, it affects both the biologic life of the tumor and the prognosis of the patient. The HER2 alteration does not appear to change over time. When it is amplified in the primary tumor, the degree of amplification seems to hold steady throughout the course of disease and is similarly amplified in subsequent metastases.
Moreover, the presence of multiple copies of the HER-2 gene, and the corresponding over-expression of its protein, plays a pivotal role in the rapid growth of tumor cells in 25-30 percent of breast cancer patients. HER2, thus, has a profound significance on breast cancer characterization and management. In a state of overexpression, its impact extends from its tumorigenicity, metastatic potential, effects on hormone dependence, and effects on response to tamoxifen and chemotherapy. Determination of HER-2 status is therefore a critical tool for selecting appropriate therapeutic options. The detection of amplification of the HER2 gene, as a measure of patient disease status and survivability, is further described in U.S. Pat. No. 4,968,603, incorporated herein by reference. In addition, a HER2 inhibitor, “Herceptin” has been developed by Genentech (South San Francisco, Calif.).
Other molecular differences in breast tissue have also been found to be significant in the diagnosis, characterization, management, and therapy of breast cancer. For example, the large scale molecular differences between estrogen receptor (“ER”) negative and ER positive tumors are particularly interesting in the light of clinical observations which indicate that the natural history and biological behavior of ER positive and ER negative tumors are distinct even in the absence of hormonal therapy. For example, ER negative cancers tend to recur sooner and show a different rate of recurrence in distant organ sites compared to ER positive tumors. The clinical observations and the molecular profiling data emerging from several laboratories, now strongly suggest that ER negative and ER positive breast cancers are two distinct disease entities rather than phenotypic variations of the same disease. (Pusztai, L. et al., in publication).
If the clinically significant determination is the status of the ER pathways, not just the status of the ER itself, then measurements of gene expression profiles that reflect activity of the pathway could also provide an important advance in understanding the behavior of breast cancers. Genes that encode proteins that synergize with ER, such as HNF3 antibody and androgen receptor, may also provide significant information regarding breast cancers.